Optimal combination of anti-inflammatory components from Chinese medicinal formula Liang-Ge-San.

ETHNOPHARMACOLOGICAL RELEVANCE: Liang-Ge-San (LGS), a traditional Chinese medicine (TCM) formula, is usually used in acute inflammatory diseases in China. AIM OF THE STUDY: This study aims to detect the optimal combination of anti-inflammatory components from LGS. MATERIALS AND METHODS: Four mainly representative components (phillyrin, emodin, baicalin, and liquiritin) from LGS were chosen. The optimal combination was investigated by orthogonal design study. Zebrafish inflammation model was established by lipopolysaccharide (LPS)-yolk microinjection, and then the anti-inflammatory activities of different combinations were determined by survival analysis, changes on inflammatory cells infiltration, the MyD88/NF-κB and MAPK pathways and inflammatory cytokines production. RESULTS: The different combinations of bioactive ingredients from LGS significantly protected zebrafish from LPS-induced inflammation, as evidenced by decreased recruitment of macrophages and neutrophils, inhibition of the MyD88/NF-κB and MAPK pathways and down-regulation of TNF-α and IL-6. Among them, the combination group 8 most significantly protected against LPS. The combination of group 8 is: 0.1 µM of emodin, 2 µM of baicalin, 20 µM of phillyrin and 12.5 µM of liquiritin. CONCLUSION: The optimized combination group 8 exerts the most significant anti-inflammatory activity by inhibiting the recruitment of inflammatory cells, activation of the MyD88/NF-κB and MAPK pathways and the secretion of pro-inflammatory cytokines. This present study provides pharmacological evidences for the further development of new modern Chinese drug from LGS to treat acute inflammatory diseases, but indicated the use of zebrafish in the screening of components from formulas.

Ainsliaea fragrans champ. Extract prevents cervicitis in BALB/c mice and regulates MyD88-NF-κB signaling pathway in MALP-2-stimulated RAW264.7 cells.

Ethnopharmacological relevance Ainsliaea fragrans Champ. (A. fragrans) is used to treat infection of the lower genital tract in gynecology, such as cervicitis and pelvic inflammatory disease. This study analyzed the therapeutic efficiency of A. fragrans on cervicitis and the inhibition mechanism of AF-p2 in MALP-2-stimulated RAW264.7 cells. Materials and methods The anti- Ureaplasma urealyticum (Uu) activity of A. fragrans and AF-p2 were determined by antimicrobial susceptibility testing. The activity of A. fragrans extracts (AFext) was evaluated in female BALB/c mice with cervicitis induced by Uu. Furthermore, the therapeutic mechanism of AFext and AF-p2 on myeloid differentiation factor 88 (MyD88) pathway were studied in macrophage activating lipopeptide-2 (MALP-2) irritated RAW264.7 cells. Results AFext could suppress the proliferation of Uu in vitro, including the azithromycin resistant strains. Meanwhile, AFext prevented cervicitis caused by Uu infection in BALB/c mice. Moreover, both AFext and AF-p2 could significantly suppress the nitric oxide (NO) production as well as other proinflammatory cytokines (IL-1ß,IL-6,TNF-α) in MALP-2 stimulated RAW264.7 cells. Moreover, AF-p2 also down-regulated iNOS, p65, Iκ-Bα, MyD88 and cyclooxygenase-2 (COX-2) levels in RAW264.7 cells. Conclusion This study indicated that AFext had a therapeutic effect in cervicitis induced by Uu infection. Furthermore, the lead compound AF-p2 showed an anti-infectious effect in MALP-2 irritated RAW264.7 cells through downregulating MyD88-NF-κB signaling pathway.

Forsythiae Fructuse water extract attenuates liver fibrosis via TLR4/MyD88/NF-κB and TGF-ß/smads signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Forsythiae Fructuse water extract (FSE) is a water-soluble component extracted from the traditional Chinese medicine Forsythiae Fructuse (The fruit of Forsythia suspensa (Thunb.) Vahl) usually used to treat inflammatory diseases. However, little is known about the therapeutic effect of FSE on liver fibrosis. AIM OF THE STUDY: The purpose of our study was to investigate the therapeutic effect of FSE on liver fibrosis and reveal the underlying mechanism. MATERIALS AND METHODS: Liver fibrosis model was established by subcutaneous injection of olive oil containing 40% CCl4. Rat liver tissue morphologic pathology was investigated by using HE staining, Masson staining and Sirius red staining. Several biochemical markers including liver (ALT, AST, AKP, γ-GT), fibrosis (HA, LN, PC III, Col IV) and inflammation (IL-6, IL-1ß, TNF-α) were determined by using Elisa kits. Immunohistochemistry was used to observe the distribution of α-SMA and COL1 in liver tissue. Effects of FSE on inflammatory pathway (TLR4/MyD88/NF-κB) and fibrotic pathway (TGF-ß/smads) were detected by western blot and qPCR. RESULTS: The results showed that hepatic histopathological injury, abnormal liver function, fibrosis and inflammation induced by CCl4 were improved by FSE (2.5, 5 g/kg). Immunohistochemistry and western blot results indicated that the expression of α-SMA and COL1 in liver tissue was inhibited by FSE (2.5, 5 g/kg). Western blot and qPCR results further proved that FSE (2.5, 5 g/kg) inhibited the transduction of TLR4/MyD88/NF-κB and TGF-ß/smads signaling pathways. CONCLUSION: FSE can inhibit the expression of inflammatory factors and fibrotic cytokines, reduce liver injury, and inhibit the development of liver fibrosis through TLR4/MyD88/NF-κB and TGF-ß/smads signaling pathways.

Danshen (Salvia miltiorrhiza) restricts MD2/TLR4-MyD88 complex formation and signalling in acute myocardial infarction-induced heart failure.

Heart failure (HF) represents a major public health burden. Inflammation has been shown to be a critical factor in the progression of HF, regardless of the aetiology. Disappointingly, the majority of clinical trials targeting aspects of inflammation in patients with HF have been largely negative. Many clinical researches demonstrate that danshen has a good efficacy on HF, and however, whether danshen exerts anti-inflammatory effects against HF remains unclear. In our study, the employment of a water extracted and alcohol precipitated of danshen extract attenuated cardiac dysfunction and inflammation response in acute myocardial infarction-induced HF rats. Transcriptome technique and validation results revealed that TLR4 signalling pathway was involved in the anti-inflammation effects of danshen. In vitro, danshen reduced the release of inflammatory mediators in LPS-stimulated RAW264.7 macrophage cells. Besides, the LPS-stimulated macrophage conditioned media was applied to induce cardiac H9C2 cells injury, which could be attenuated by danshen. Furtherly, knock-down and overexpression of TLR4 were utilized to confirm that danshen ameliorated inflammatory injury via MyD88-dependent TLR4-TRAF6-NF-κB signalling pathway in cardiomyocytes. Furthermore, by utilizing co-immunoprecipitation, danshen was proved to suppress MD2/TLR4 complex formation and MyD88 recruitment. In conclusion, our results demonstrated that danshen ameliorates inflammatory injury by controlling MD2/TLR4-MyD88 complex formation and TLR4-TRAF6-NF-κB signalling pathway in acute myocardial infarction-induced HF.

Protective effects of syringin against oxidative stress and inflammation in diabetic pregnant rats via TLR4/MyD88/NF-κB signaling pathway.

Gestational diabetes (GDM) is common in pregnancies due to the inflammation and oxidative stress-mediated insulin resistance. In the present study, GDM was induced in the Wistar rats by administering the streptozotocin to elucidate whether the administration of syringin (50 mg/kg/day) during pregnancy could improve maternal glycemia and protect against the complications of GDM. The animals were assessed for their morphological changes in the ß-islets of Langerhans and their insulin-producing ability, inflammatory cytokine markers, and the involvement of TLR4/MyD88/NF-κB signaling pathway using RT-PCR. The results demonstrated that the onset of GDM demonstrated pancreatic tissue degeneration in the islets of Langerhans with a significant increase in oxidative stress and reduced antioxidant enzymes. Besides, the mRNA expression levels of TLR4, MyD88, NF-Kß p65; NLRP3 mRNA were profoundly increased in GDM rats compared to normal pregnant rats. On the other hand, syringin administered GDM rats abrogated the oxidative stress and attenuated the level of the inflammatory cytokines. Intriguingly, the decrease in TLR4 expression and the downstream molecules of MyD88, NF-κB, and NLRP3 were also observed in syringin administered GDM rats that indicate the insulin secretion stimulatory actions of syringin through the suppression of TLR4 signaling. These novel findings of the study provide evidence that syringin could be a probable candidate to be used in the treatment of gestational diabetes in the future.

Electroacupuncture Alleviates Paclitaxel-Induced Peripheral Neuropathic Pain in Rats via Suppressing TLR4 Signaling and TRPV1 Upregulation in Sensory Neurons.

Paclitaxel-induced peripheral neuropathy is a common adverse effect during paclitaxel treatment resulting in sensory abnormalities and neuropathic pain during chemotherapy and in cancer survivors. Conventional therapies are usually ineffective and possess adverse effects. Here, we examined the effects of electroacupuncture (EA) on a rat model of paclitaxel-induced neuropathic pain and related mechanisms. EA robustly and persistently alleviated paclitaxel-induced pain hypersensitivities. Mechanistically, TLR4 (Toll-Like Receptor 4) and downstream signaling MyD88 (Myeloid Differentiation Primary Response 88) and TRPV1 (Transient Receptor Potential Vallinoid 1) were upregulated in dorsal root ganglion (DRGs) of paclitaxel-treated rats, whereas EA reduced their overexpression. Ca2+ imaging further indicated that TRPV1 channel activity was enhanced in DRG neurons of paclitaxel-treated rats whereas EA suppressed the enhanced TRPV1 channel activity. Pharmacological blocking of TRPV1 mimics the analgesic effects of EA on the pain hypersensitivities, whereas capsaicin reversed EA's effect. Spinal astrocytes and microglia were activated in paclitaxel-treated rats, whereas EA reduced the activation. These results demonstrated that EA alleviates paclitaxel-induced peripheral neuropathic pain via mechanisms possibly involving suppressing TLR4 signaling and TRPV1 upregulation in DRG neurons, which further result in reduced spinal glia activation. Our work supports EA as a potential alternative therapy for paclitaxel-induced neuropathic pain.

Posttreatment With LYRM03 Protects Rats From Acute Lung Inflammation Induced by Lipopolysaccharide via Suppressing the NF-κB/MyD88/TLR4 Axis.

BACKGROUND: 3-amino-2-hydroxy-4-phenyl-valyl-isoleucine (LYRM03) has been shown to be beneficial in a rat model of acute lung injury (ALI). Nonetheless, the pharmacologic action of LYRM03 interference has not been demonstrated to occur through oxidative stress and apoptosis in a rat lipopolysaccharide (LPS)-induced ALI model, and the potential pathogenic mechanism needs to be clarified. Our research intended to explore the mechanism of action using an in vivo rat LPS-induced ALI model and highlight the associated pathogenesis. METHODS: Sprague-Dawley rats were randomly assigned to the following five groups: Sham; LPS (5 mg/kg); LPS + LYRM03 (5 mg/kg); LPS + LYRM03 (10 mg/kg); and LPS + LYRM03 (20 mg/kg). Pulmonary injury indicators were documented at 24 h after LPS-induced ALI. Morphologic alterations, such as the extent of the injury, were determined using hematoxylin-eosin staining. Furthermore, expression levels of oxidative stress indicators (malondialdehyde, superoxide dismutase, and glutathione peroxidase) and inflammatory molecules (tumor necrosis factor-α, interleukin-8, and interleukin-6) in circulation were observed. The production of apoptosis-associated proteins (poly ADP-ribose polymerase, c-caspase 3, B-cell lymphoma-2, and Bcl2 associated X), inflammatory mediators (high mobility group box-1, toll-like receptor 4, nuclear factor-kappa B p65, and myeloid differentiation primary response 88), and inhibitor of kappa B-α were determined through Western blotting. Real-time polymerase chain reaction was applied to assess the messenger RNA expression of the inflammatory mediators. RESULTS: The LPS-treated group exhibited a remarkable increase in the extent of the pulmonary injury, oxidative stress indicator secretion, inflammatory molecule release, and inflammatory mediator production and an increase in the inhibitor of kappa B-α levels relative to the Sham group. The LYRM03 (5 and 10 mg/kg)-treated groups exhibited a remarkable decrease relative to the LPS group. In addition, treatment with LYRM03 (20 mg/kg) powerfully limited the extent of the injury and demonstrated anti-inflammatory actions. CONCLUSIONS: The results of this investigation indicated that treatment with LYRM03 plays a role in lung defense by inhibiting the NF-κB/MyD88/TLR4 pathway.

Icariside II attenuates lipopolysaccharide-induced neuroinflammation through inhibiting TLR4/MyD88/NF-κB pathway in rats.

Inflammation in central nervous system (CNS) plays a vital role in neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Lewy body dementia (DLB), HIV-related dementia and traumatic brain injury. Icariside II (ICS II), an active flavonoid compound derived from a Chinese herbal medicine Epimedium brevicornum Maxim, has been shown to possess a neuroprotective effect on AD model. However, whether ICS II has a directly protective effect on acute neuroinflammation remains still unclear. Therefore, the current study was designed to investigate the possible protective effect of ICS II on acute neuroinflammation induced by intracerebroventricular (ICV) injection of lipopolysaccharide (LPS), and further to explore its possible mechanism. After ICS II was prophylactically administered for 7 days before LPS injection, the rats were randomly divided into five groups as follows: sham group (n = 9), sham + ICS II-H (10 mg/kg) (n = 9), LPS (n = 14), LPS + ICS II-L (3 mg/kg) (n = 14), LPS + ICS II-H (10 mg/kg) (n = 14) groups, respectively. As expected, LPS injection exhibited neuronal morphological damage, and ionized calcium binding adapter molecule 1 (IBA-1) of microglia and glial fibrillary acidic protein (GFAP) of astrocyte were activated. However, pre-treatment with ICS II not only inhibited the activation of microglia and astrocyte, but also significantly reversed the expressions of inflammatory factors such as interleukin-1ß (IL-1ß), tumor necrosis factor (TNF-α), cyclooxygenase-2 (COX-2), as well as the expressions of Toll-Like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and TNF receptor associated factor 6 (TRAF6). Furthermore, ICS II inhibited the degradation of IκB and the following activation of NF-κB. Hence it is concluded that ICS II attenuates LPS-induced neuroinflammation through inhibiting TLR4/MyD88/NF-κB pathway in rats, and it has potential value as a new therapeutic agent to treat neuroinflammation-related diseases, such as AD.

Simultaneous targeting of MyD88 and Nur77 as an effective approach for the treatment of inflammatory diseases.

Myeloid differentiation primary response protein 88 (MyD88) has long been considered a central player in the inflammatory pathway. Recent studies clearly suggest that it is an important therapeutic target in inflammation. On the other hand, a recent study on the interaction between the orphan nuclear receptor (Nur77) and p38α, leading to increased lipopolysaccharide-induced hyperinflammatory response, suggests this binary complex as a therapeutic target. In this study, we have designed inhibitors that can inhibit both MyD88 and Nur77 at the same time. Since both MyD88 and Nur77 are an integral part of the pathways involving lipopolysaccharide-induced activation of NF-κB-mediated inflammation, we tried to target both proteins with the same library in order to retrieve compounds having dual inhibitory properties. To perform this, we developed a homodimeric model of MyD88 and, along with the crystal structure of Nur77, screened a virtual library of compounds from the traditional Chinese medicine database containing ~61,000 compounds. We analyzed the resulting hits for their efficacy for dual binding and probed them for developing a common pharmacophore model that could be used as a prototype to screen compound libraries as well as to guide combinatorial library design to search for ideal dual-target inhibitors. Thus, our study explores the identification of novel leads having dual inhibiting effects due to binding to both MyD88 and Nur77 targets.

Wuling San protects kidney dysfunction by inhibiting renal TLR4/MyD88 signaling and NLRP3 inflammasome activation in high fructose-induced hyperuricemic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Wuling San, a famous prescription in Chinese medicine, is composed of Polyporus, Poria, Alismatis rhizoma, Cinnamomi cortex and Atractylodis macrocephalae rhizoma, and promotes kidney function and diuresis. The main purpose of this study was to investigate its renal protective effect in high fructose-induced hyperuricemic mice. MATERIALS AND METHODS: ICR mice were fed with 30% fructose in drinking water for 6 weeks to induce hyperuricemia and renal dysfunction. Then mice were orally administrated for other 6 weeks with Wuling San (987, 1316, 1755 and 2340mg/kg), allopurinol (5mg/kg) and water daily, respectively. Serum and urine levels of uric acid, creatinine and blood urea nitrogen (BUN) were measured. Hematoxylin and eosin staining was used to assess renal histological changes. Renal interleukin (IL)-1ß concentrations were measured using ELISA kit. Renal protein levels of organic ion transporters, as well as toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling and pyrin domain containing 3 (NLRP3) inflammasome were determined by Western blot assay. RESULTS: Wuling San significantly decreased serum uric acid, creatinine and BUN levels, increased fractional excretion of uric acid (FEUA) in fructose-fed mice. It restored fructose-induced dysregulation of renal urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), ATP-binding cassette subfamily G member 2 (ABCG2) and organic anion transporter 1 (OAT1), as well as organic cation transporter 1 (OCT1) and OCT2 in mice. Wuling San obviously alleviated infiltration of inflammation cells in kidney glomerulus of fructose-fed mice. Moreover, Wuling San suppressed the activation of TLR4/ MyD88 signaling to inhibit nuclear factor κB (NF-κB) signaling and mitogen-activated protein kinases (MAPKs) activation in fructose-fed mice. Additionally, Wuling San decreased NLRP3 inflammasome activation and IL-1ß secretion in the kidney of fructose-fed mice. CONCLUSION: Wuling San exerts renal protective effect by modulating renal organic ion transporters in fructose-induced hyperuricemic mice. The molecular mechanism of its action may be associated with the suppression of TLR4/MyD88 signaling and NLRP3 inflammasome activation to reduce IL-1ß production in high fructose-induced hyperuricemic mice.

The impact of membrane lipid composition on macrophage activation in the immune defense against Rhodococcus equi and Pseudomonas aeruginosa.

Nutritional fatty acids are known to have an impact on membrane lipid composition of body cells, including cells of the immune system, thus providing a link between dietary fatty acid uptake, inflammation and immunity. In this study we reveal the significance of macrophage membrane lipid composition on gene expression and cytokine synthesis thereby highlighting signal transduction processes, macrophage activation as well as macrophage defense mechanisms. Using RAW264.7 macrophages as a model system, we identified polyunsaturated fatty acids (PUFA) of both the n-3 and the n-6 family to down-regulate the synthesis of: (i) the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α; (ii) the co-stimulatory molecule CD86; as well as (iii) the antimicrobial polypeptide lysozyme. The action of the fatty acids partially depended on the activation status of the macrophages. It is particularly important to note that the anti-inflammatory action of the PUFA could also be seen in case of infection of RAW264.7 with viable microorganisms of the genera R. equi and P. aeruginosa. In summary, our data provide strong evidence that PUFA from both the n-3 and the n-6 family down-regulate inflammation processes in context of chronic infections caused by persistent pathogens.

Triptolide ameliorates Crohn's colitis is associated with inhibition of TLRs/NF-κB signaling pathway.

Growing evidence suggests that TLRs/NF-κB signaling pathway plays a critical role in the pathogenesis of Crohn's disease (CD). We have reported that triptolide, an active component from Tripterygium wilfordii Hook, showed therapeutic activity in IL-10-deficeint (IL-10-/- mice, a murine CD model. However the full mechanisms of action of this agent in CD remain largely unknown. We hypothesized that triptolide would ameliorate the experimental colitis by inhibiting TLRs/NF-κB signaling pathway. We found TLR2 and TLR4 were upregulated in IL-10-)/- mice, triptolide inhibited the TLRs/NF-κB signaling pathway in vivo. In addition, triptolide in vitro was able to downregulate the TLRs/NF-κB pathway in cultured colonic explants from CD patients. Our results confirm the therapeutic effect of triptolide in experimental colitis, and suggest it as a promising compound for CD treatment. These findings also support the possibility that targeted inhibition of TLR signaling pathway is an approach deserving further investigation as a therapeutic strategy for CD.